KLK-Targeted Therapies for Prostate Cancer

KLK-targeted therapies for prostate cancer Hannu Koistinen, Johanna Mattsson, Ulf-Håkan Stenman Department of Clinical Chemistry, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland

ARTICLE INFO ABSTRACT

Address correspondence to: Alternative treatments are urgently needed for prostate Dr Hannu Koistinen cancer, especially to address the aggressive metastatic cas- Department of Clinical Chemistry, Biomedicum Helsinki, tration-resistant disease. Proteolytic enzymes are involved P.O. Box 63, FIN-00014 in cancer growth and progression. The prostate produces University of Helsinki, Finland several proteases, the most abundant ones being two mem- Tel: +358 9 471 71734 e-mail: [email protected] bers of the kallikrein-related peptidase (KLK) family, prostate-specific antigen (PSA) and KLK2. Despite the wide use E-mails: [email protected] (HK) of PSA as a clinical marker, the function(s) of PSA and other [email protected] (JM) KLKs in prostate cancer are poorly known. Hypothetic roles [email protected] (UHS) of KLKs in prostate cancer include activities that may both Key words: promote and inhibit cancer growth and metastasis, includ- PSA, KLK3, hK2, KLK2, kallikrein, ing the antiangiogenic activity of PSA. Thus it may be pos- prostate cancer, peptide, therapy, sible to control prostate cancer growth by modulating the prodrug, serpin, vaccine proteolytic activities of KLKs. PSA and KLK2 are especially attractive targets for prostate cancer treatment because of KLK-targeted therapies for prostate cancer Hannu Koistinen, Johanna Mattsson,their proposed Ulf-Håkan roles Stenman in tumor development and inhibition of angiogenesis in combination with their prostate selective expression. So far the number of molecules affecting selectively the activity of KLKs is limited and none of these are used to treat prostate cancer. Prodrugs that, after cleavage of the peptide part by PSA or KLK2, release active drug molecules, and PSA-targeted therapeutic vaccines have already been tested clinically in humans and the first results have been encouraging. Although KLKs are attractive targets for prostate cancer treatment, much remains to be done before their potential can be fully elucidated. The objective of this review is to address the current state of the KLKs as novel therapeutic targets for prostate cancer treatment.

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INTRODUCTION PSA as a clinical marker, the function(s) of PSA and other KLKs in prostate cancer are poorly Prostate cancer is a considerable health care known [7]. While several KLKs may be involved problem. With 900,000 new cases and about in prostate cancer development, efforts to tar- 260,000 deaths worldwide in 2008, it is the get them for treatment of prostate cancer have second most frequently diagnosed cancer and concerned the two major proteases produced the sixth most common cause of cancer death in prostate, i.e., PSA and KLK2. Thus this review in men [1]. In the UK, the lifetime risk of de- will focus mainly on these KLKs and their use as veloping prostate cancer is estimated to be 1 novel therapeutic targets for prostate cancer in 8 (Cancer Research UK). Since the approval of prostate-specific antigen (PSA or kallikrein- treatment. related peptidase-3, KLK3) test by FDA in 1986, PSA has been the most widely used cancer PROTEASES IN CANCER marker [2]. However, extensive screening with Since the discovery of the role of proteases in PSA has lead to detection and unnecessary food degradation, proteases have been found treatment of cancers that would not have sur- to be involved in almost all biological pathways faced clinically without screening [2]. Prostate and networks, performing several essential cancer often presents as a multi-focal tumor functions in all living organisms, from fertiliza- with various degrees of aggressiveness. Be- tion and development to normal physiology cause of the widespread use of screening, most [8,9]. Proteases (also called peptidases or pro- prostate cancers are presently detected at an teolytic enzymes) may exhibit highly selective early stage and have favorable prognosis. While substrate cleavage or have broader specificity. most patients can be cured by radical prostatec- About 600 human proteases, which are collec- tomy or radiotherapy, these are associated with tively called the degradome, representing ~2 % side effects, and about one third of the tumors of the whole human genome are known [10]. relapse [3]. Patients can be treated by andro- Among the serine proteases, KLKs form a family gen deprivation, but eventually most of them, of 15 trypsin- and chymotrypsin-like proteases and 10-20% within 5 years, become resistant to [11,12]. Protease activity is controlled by sever- this therapy, i.e., develop castration-resistant al mechanisms, including regulation of gene ex- prostate cancer (CRPC) [4]. Currently there is no pression, activation of their inactive pro-forms cure for these cancers [5]. While CRPCs respond (zymogens) either autocatalytically or by other to some treatment modalities, the effect on proteases, inhibition of their activity by endog- survival is generally modest, i.e., some months. enous protease inhibitors, and phosphorylation Therefore, it is important to develop alternative [8,13]. Many proteases, including those of the treatments that are either curative, prevent the KLK family, act in cascades or networks, which development of CRPC and/or formation of met- facilitates signal amplification and stringent reg- astatic lesions that eventually kill the patients, ulation of their activity [14]. or to slow down the growth of small tumors, in order to prevent them from surfacing clinically Alterations in proteolytic systems underlie sev- within the lifetime of the patient. Proteolytic eral pathological conditions, including cancer, enzymes (proteases), including 15 members and proteases have been found to play a signifi- of the kallikrein-related peptidase (KLK) fam- cant role at virtually all stages of tumor progres- ily, are potential targets for treatment of pros- sion [9]. The roles of proteases in cancer have tate cancer [6]. Despite the widespread use of been widely studied since the discovery of their

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role in cancer cell invasion, which is a prereq- [21]. The clinical use of PSA determinations has uisite for tumor invasion and metastasis forma- been reviewed in several recent articles [22,23]. tion. In addition to degrading extracellular ma- Among the KLKs, KLK2 has attracted most inter- trix proteins and adhesion molecules facilitating est after PSA, due to its prostate specificity and cell invasion, proteases have several other func- relatively high expression levels [18]. Contrary tions relevant for cancer, including activation of to PSA, KLK2 expression in prostate cancer is protease-activated receptors (PARs) and regula- higher than in benign prostate [23,24]. The ra- tion of the activity of other signaling molecules, tio between hK2 and PSA mRNA increases with like kinases and growth factors [8,13,15]. Can- increasing grade [25]. cer has been thought to be primarily associated with increased proteolytic activity and while Noteworthy, single nucleotide polymorphisms this is true for many proteases, some proteases (SNPs) in KLK genes have been shown to be asso- exert opposite effects, such as acting as tumor ciated with prostate cancer [26]. Some of these suppressors by suppressing angiogenesis or in- SNPs affect the expression levels of KLKs. How- ducing apoptosis [16,17]. ever, apart from these genetic polymorphisms and hormonal regulation [11], the mechanisms Expression of KLKs in prostate cancer behind the altered regulation of KLK expression in tumors still remains largely unsolved. The prostate produces several proteases, the most abundant ones being two KLKs, PSA and Functions of KLKs in prostate cancer KLK2 [18]. Shaw and Diamandis reported that all 15 KLKs are expressed in the prostate at the The suggested functions of KLKs include both mRNA level [18]. In tissue extracts they found, those that promote and inhibit tumor growth in addition to KLK2 and PSA, KLK1, -4, -5, -9, -11, and metastasis [7,11,12,27]. The physiological -13, -14 and -15. PSA is expressed in differenti- function of PSA, and perhaps also other pros- ated luminal epithelial cells of the prostate and tatic KLKs, is to promote sperm motility by dis- secreted into seminal fluid. The levels in extra- solving the seminal clot formed after ejacula- cellular fluid of the prostate are up to 2 µM or in tion by cleaving semenogelins. KLKs are also prostate tissue 10 mg/g of tissue [18,19]. Most able to cleave several prostate cancer related of this PSA is enzymatically active [19]. Howev- substrates, at least in vitro [11]. However, hy- er, when active PSA and other KLKs reach cir- pothetic functions based on in vitro cleavage culation, they are rapidly inactivated by prote- should be interpreted with caution. In clinical ase inhibitors that are present in vast excess in studies, low PSA levels in prostate cancer tis- circulation. While PSA is a major constituent of sue are associated with poor prognosis [20,28], seminal fluid, only a minor part of it leaks out while high PSA levels are associated with low into circulation. Interestingly, the tissue con- blood vessel density [29,30]. However, the centrations of PSA are lower in malignant than PSA concentrations in serum are sometimes in normal prostatic epithelium and they are increased decades before the development of further reduced in poorly differentiated (high otherwise detectable tumors [31,32]. This sug- Gleason grade) tumors [20]. In spite of this, PSA gests that PSA may initiate or facilitate early is the best cancer marker presently available cancer development. [2,7]. This is based on increased leakage into Cancer cells have to acquire several biological circulation from malignant prostatic tissue that capabilities during the multistep development has lost connection with the prostatic ducts of tumors described by Hanahan and Weinberg

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in Hallmarks of Cancer [33]. Several described the dependence on enzymatic activity is con- or hypothesized functions of KLKs are relevant troversial [38]. However, our studies strongly for these effects [7]. The ability to proliferate suggest that PSA activity is needed for the an- and evade growth-suppressing signals is one of tiangiogenic activity, as the enzymatic activity the essential properties of cancer cells. Several of different PSA forms present in seminal fluid studies suggest that PSA and other KLKs may correlates with the antiangiogenic activity [39]. promote the growth of prostate cancer by stim- Furthermore, inhibition of PSA by small mole- ulating cell proliferation [34,35]. Furthermore, cule inhibitors or an antibody abolishes the an- PSA has been found to promote the growth of tiangiogenic activity [40], while the stimulation prostate cancer xenograft tumors [34]. In con- of PSA activity by peptides enhanced it [41]. trast to these studies, Bindukumar et al. [37] Several KLKs, like PSA and KLK2, are able to de- found that subcutaneously administered PSA grade extracellular matrix proteins and activate reduced the growth of xenograft tumors in other extracellular matrix degrading proteases mice. Several KLKs have been found to activate or inactivate their inhibitors [11,14]. These stud- growth-factors and PARs [11,15], which lead to ies suggest that KLKs are involved in proteolytic a wide array of responses, including promotion cascades facilitating prostate cancer growth of cancer cell growth and invasion. In addition and metastasis [14]. Indeed, PSA-treatment has to increasing cell proliferation, PSA has been been found to increase invasion of prostate can- shown to reduce apoptosis [35], which is also cer cells in vitro [42]. Other studies suggest that essential for cancer development. PSA may play a role in the development of bone Like all tissues, tumor needs nutrients and oxy- metastases (reviewed in [11,43]). gen and ability to remove waste and carbon Knockout studies of PSA or KLK2 have not been dioxide in order to grow and survive [33]. This performed as mice and other animals used for requires vascularization and thus tumors need such studies do not have genes encoding PSA to develop new blood vessels in order to grow or KLK2 [11]. Most studies aiming to solve the beyond a size of 2-3 mm3 [37]. Prostate cancer functions of KLK2, PSA, and other KLKs have grows unusually slowly after reaching this size, utilized cancer cell lines. However, the in vitro which corresponds to the time when it can be growth characteristics of these cells may not detected by prostate biopsy of men with elevat- necessarily predict tumorigenicity and different ed serum concentrations of PSA [31]. The slow cell lines may show very different responses [7]. growth of prostate cancer could be dependent Furthermore, cancer cells grown in an isolated on the antiangiogenic activity of PSA. Several environment behave very differently from those studies have addressed the antiangiogenic role in tumors and in contact with extracellular ma- of PSA, which has been demonstrated in cell trix and stromal cells [44]. Transgenic mice ex- culture models at sub-physiological PSA con- pressing PSA and/or KLK2 in the prostate have centrations [16,38,39]. In a pioneering study by Fortier et al., PSA was shown to inhibit en- been developed. In these, neither PSA nor KLK2 dothelial cell tube formation, growth, invasion have been found to initiate cancer or cause any and migration [16]. They further showed that morphological changes [45]. However, the PSA subcutaneous administration of PSA inhibits levels in these are about 1000-fold lower than angiogenesis in an in vivo model of blood ves- those in the human prostate. sel growth [38]. The mechanism by which PSA Taken together, these studies suggest that PSA exerts its antiangiogenic effect is unclear. Even and other KLKs may affect tumor growth and

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perhaps even initiate cancer development. However, these are rather non-specific inhibi- The effects of KLKs may be different at- differ tors. So far the number of molecules affect- ent stages of tumor growth, e.g., PSA may favor ing selectively the activity of individual KLKs tumor development at early stages of cancer, is very limited and none of these are used to for example by activating growth factors, but at treat prostate cancer [6,49,52]. For PSA, both later stages it may inhibit tumor growth by its activity-stimulating and inhibiting compounds antiangiogenic activity [7]. have been developed, while for KLK2 and KLK4 only inhibitors have been described (Table). KLK-TARGETED THERAPIES Prodrugs that are activated by PSA or KLK2, and FOR PROSTATE CANCER PSA-targeted therapeutic vaccines have already been tested clinically in humans and prelimi- Recently, proteases have been estimated to nary results are encouraging [53,54] (Table). represent 5-10% of the potential drug targets [46,47] but the number of new approved prote- PSA inhibitors and stimulators ase inhibitors is still limited [48]. A problem with Several peptide-based or small molecule inhibi- proteases as drug targets is the lack of specific- tors for PSA have been described (reviewed in ity. Thus, inhibitors tend to react with similar [6,27,49,52]). These have been found either by proteases affecting a broad range of protease high-throughput screening or by rational drug activities that are crucial for normal physiology. design. Several antibodies that inhibit or stimu- For example, lack of specificity was a major rea- late PSA activity have also been described [55]. sons for the failure of early matrix metallopro- Some of these inhibitors have been tested in tease (MMP)-inhibitors, as they also inhibited prostate cancer relevant models and found to MMPs that are needed for normal tissue func- inhibit antiangiogenic activity of PSA in a cell tion, or act as tumor suppressors [46,47,17]. culture model [40], inhibit the growth of pros- PSA and KLK2 are attractive targets for prostate cancer cell lines or exert a small inhibitory tate cancer treatment because of their possible effect on xenograft tumor growth [56]. It should roles in tumor development, metastasis and in- be noted that the specificity of these inhibitors hibition of angiogenesis, and their prostate se- has not been thoroughly characterized. lective expression, which together with the lack Since PSA shows antiangiogenic activity and, of active forms in circulation makes systemic thus could inhibit development of prostatic effects unlikely. Furthermore, as their proposed tumors, we have been interested in molecules physiological function is related to liquefaction that stimulate the activity of PSA. By screening of the seminal fluid clot, their targeting is not of almost 50,000 drug-like small molecules, we likely to cause severe side-effects other than found some PSA inhibitors but did not identi- those related to fertility. In addition to PSA and fy compounds that stimulate PSA activity [40]. KLK2, KLK4 is also a potential target for prostate While small molecule drugs have several advan- cancer treatment [49]. KLK4 is overexpressed in tages as compared to peptides, their specificity prostate cancer, promotes cell proliferation and for similar proteases is more limited. Further- cleaves several cancer associated substrates, in- more, only 30-50% of the targets that represent cluding PARs [11,50,51]. an opportunity for therapeutic intervention Several naturally occurring proteins, includ- have been suggested to be amenable to tradi- ing serpins, Kazal-type serine protease inhibi- tional small molecule approaches [57]. Thus, we tors and α2-macroglobulin, inhibit KLKs [49,52]. have used phage-display to develop peptides,

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Table Summary of the major findings on different KLK targeted therapies for prostate cancer

Strategy/target Agent Major outcome/therapeutic effecta References

Inhibitors

Inhibit prostate cancer cell and xenograft small molecules or 6,27,40,49, PSA tumor growth; inhibit antiangiogenic peptide based 52,56 activity of PSA modifed serpinb, KLK2 Reduce xenograft tumor growthb 63-66 peptide KLK4 peptide Not determined 68,69 Stimulators

peptide, small Stimulate antiangiogenic activity of PSA in PSA 58-62 molecule cell model Prodrugs

Selectively kills PSA-producing cells in vitro; selective antitumor effect on PSA-producing PSA substrate tumor xenografts in mice and monkeys; PSA combined with a 73-84 significant improvement of symptoms in toxic drug molecule patients with benign prostatic hyperplasia with only mild, locally limited side effects

Significant antitumor effect in tumor KLK2 substrate xenografts in vivo, but prolonged KLK2 combined with a administration caused local toxic effect; 85 toxic drug molecule less effective than similar PSA-activated prodrug Vaccination

antigen (PSA), DNA- Safe in phase I and II studies, showing based vaccines, PSA prostate specific T lymphocyte responses 53-54 usually include and benefit for some of the patients other antigens a The mentioned outcomes and therapeutic effects may be valid only for some of the agents for a given strategy and target. b The agent, modified serpin, not specific for KLK2.

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which stimulate the activity of PSA several fold KLK5 and KLK14 [64]. This molecule, called MD- at µM concentrations [58,59]. These peptides PK67b, has been shown to reduce the growth also stimulate PSA-activity towards protein of prostate cancer xenograft tumors producing substrates but they do not affect the activity KLK2. Further clinical studies, including evalu- of several enzymatically or structurally related ation of safety in humans, have been initiated proteases (Mattsson et al., unpublished data) [64]. [59]. The use of peptides for drug discovery is As our phage-display approach was successful a rapidly emerging field. The pharmacokinetic with PSA, we also developed peptide inhibi- and other properties of peptides can be modi- tors for KLK2 using this approach [65]. While fied and they can serve as starting structures all of the PSA-stimulating peptides were cyclic for development of peptidomimetics. We have containing one or two disulfide-bridges, all the been able to significantly improve the stabil- KLK2 inhibiting peptides were identified in lin- ity of some peptides stimulating PSA activity ear peptide libraries. The identified peptides and created the first pseudopeptides, in which inhibited KLK2 at µM concentrations. Like with parts of the peptide have been replaced by non- PSA-stimulating peptides, we have been able to peptidic structures without loss of bioactivity significantly improve the stability of the KLK2- [60,61]. We hypothesize that modified peptides inhibitory peptides [66]. or peptidomimetic compounds based on these can be used for imaging and proof of principle Sunflower trypsin inhibitor (SFTI), which is a 14 studies, and eventually for treatment of pros- amino acid residue cyclic peptide structurally tate cancer. The peptides enhance the antian- similar to Bowman-Birk family of serine prote- giogenic activity of PSA in cell culture models ase inhibitors, is a potent and broad-range pro- [41] but in preliminary animal studies, the first tease inhibitor [49,67]. Recently, SFTI has been generation peptides have not shown any major modified to selectively and efficiently inhibit effect on tumor growth (our unpublished re- KLK4, using a combination of molecular model- sults). This is not surprising as the peptides are ing and substrate screening [68], and further in quickly excreted. Using pharmacophore-based silico screening of inhibitor variants in complex virtual screening we have recently identified with KLK4 or trypsin [69]. Although promising the first small drug-like molecule that- stimu results with the SFTI-based KLK4 inhibitor have lates PSA activity [62]. been obtained using ovarian cancer models [70], its effect on prostate cancer have not yet KLK2 and KLK4 inhibitors been reported. Since many cancers, including prostate cancer, PSA- and KLK2-activated prodrugs are associated with increased activity of several proteases, inhibitors, in addition to those for Perhaps the most promising results concerning PSA, have been developed for KLK2 and KLK4 the use of KLKs in prostate cancer treatment for the targeting of prostate cancer. Cloutier et have been obtained using prodrugs that are al. have used phage-display to screen a library activated by PSA or KLK2. Protease-activated of variants of α1-anti-chymotrypsin (ACT), which prodrugs are promising for targeted delivery of inhibits several proteases. They identified a drugs into a specific tissue. The inactive prodrug modified version of ACT that showed selectiv- consists of a toxic drug molecule conjugated to ity towards KLK2 [63], but it was later found to a peptide. The prodrug is activated in the target also inhibit several other KLKs, especially KLK4, tissue through cleavage of the peptide moiety

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by a specific protease leading to release of the A thapsigargin-based prodrug has been devel- active drug molecule [71,72]. Thus, the effect of oped for KLK2 [85]. A prodrug with a KLK2 pep- the drug is directed to a specific tissue. PSA and tide substrate conjugated to the thapsigargin KLK2 are well suited as prodrug activators as analog, L12ADT, showed a significant antitumor they have highly tissue specific expression, i.e., effect in human prostate tumor xenografts in significant amounts of active PSA and KLK2 are vivo, but prolonged administration caused local found only in the prostate [6,18]. toxic effects [85]. Moreover, the antitumor -ef fect was only modest when compared to a simi- Several PSA-activated prodrugs have been lar PSA prodrug with thapsigargin. developed using peptide sequences that are highly selective for PSA [73,74]. Drug molecules PSA-targeted therapeutic vaccines conjugated to these peptides include doxorubi- Prostate cancer is considered an attractive tar- cin [73-75], vinblastine [76], 5-fluorodeoxyuri- get for development of therapeutic vaccines as dine [77], thapsigargin [78] and paclitaxel [79]. it expresses several prostate-specific proteins A doxorubicin conjugated prodrug L-377,202 and generally grows very slowly. Indeed, sev- selectively killed PSA-producing human pros- eral vaccination strategies have been estab- tate cancer cells in vitro and was 15 times more lished, some of which target PSA (reviewed in effective than conventional doxorubicin atin- [53,54]). Usually these vaccines also target oth- hibiting the growth of human prostate tumor er antigens in order to improve the immune re- xenografts in vivo [74]. Another PSA-activated sponse. For PSA, viral and DNA-based vaccines prodrug, PRX302, with the bacterial toxin pre- have been used, both encoding PSA and, usu- cursor proaerolysin, showed selective antitu- ally, other antigens. These vaccines have been mor effect on PSA-producing tumor xenografts found to be safe in phase I and II studies, show- in mice, caused extensive damage to PSA-pro- ing prostate specific T lymphocyte responses ducing prostate cells in monkeys and showed and, at least in some cases, some benefit for no toxicity in other tissues [80]. These two PSA- the patients [53,54]. It seems that these- vac cines would be especially beneficial for patients activated prodrugs have been taken to phase I with early-stage disease but results from phase and II clinical trials, where both were well toler- III studies are not available yet. ated, although L-377,202 caused neutropenia at higher doses [75]. Intraprostatic injections CONCLUSIONS of PRX302 lead to significant improvement of symptoms in patients with benign prostatic -hy PSA is an established marker for prostate can- perplasia while only mild, locally limited side ef- cer and several other KLKs are potential mark- fects were reported [81]. ers. As circulating PSA level is often used as a surrogate marker for tumor burden in preclini- Recent studies of PSA-activated prodrugs re- cal and clinical studies [73-76,80,83], and more port utilization of cell penetrating peptides to importantly to monitor relapse in patients, it deliver drugs inside prostate cancer cells [82], would be important to address whether KLK development of prodrug modifications using targeted therapies could affect PSA levels also e.g. albumin as a drug carrier [83] and synthesis otherwise than by affecting the volume of the of fusion peptides with multiple specificities to PSA producing tumor tissue. This is possible as target the drug effect to the cells that express PSA-targeted activity modulators and vaccina- specific receptors [84]. tion may affect, through several mechanisms,

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the circulating PSA levels and also the detection DISCLOSURES of PSA by immunoassay. While in prodrug stud- Ulf-Håkan Stenman holds patents for KLK-activ- ies PSA seems to be a good marker for tumor ity modulating peptides. Hannu Koistinen and burden, the therapy has also been found to in- Johanna Mattsson have nothing to disclose. duce initial leakage of PSA into circulation [81], perhaps because of tissue destruction. REFERENCES While conclusive evidence for the roles of the 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E and KLKs in prostate cancer development is still Forman D. Global cancer statistics. CA Cancer J Clin lacking, KLKs expressed in the prostate are likely 2011;61:69-90. to have functional role(s) making them poten- 2. Stenman UH, Abrahamsson PA, Aus G, Lilja H, Bangma tial targets for treatment of prostate cancer. We C, Hamdy FC, Boccon-Gibod L and Ekman P. Prognostic value of serum markers for prostate cancer. Scand J Urol have proposed that PSA promotes the growth Nephrol Suppl 2005;216:64-81. of small tumors, but may inhibit development 3. Ward JF and Moul JW. Rising prostate-specific antigen of large tumors at the stage when new blood after primary prostate cancer therapy. Nat Clin Pract Urol vessels are needed [7]. In addition to PSA and 2005;2:174-82. KLK2, some other KLKs, like KLK4, are also po- 4. Kirby M, Hirst C and Crawford ED. Characterising the tential targets for prostate cancer treatment. castration-resistant prostate cancer population: asys- tematic review. Int J Clin Pract 2011;65:1180-92. However, these KLKs have been less studied and their expression is not as restricted to the 5. Tammela TL. Endocrine prevention and treatment of prostate cancer. Mol.Cell.Endocrinol. 2012;360:59-67. prostate as that of KLK2 and PSA. Therefore de- 6. Sotiropoulou G and Pampalakis G. Targeting the kal- velopment of treatment based on these is more likrein-related peptidases for drug development Trends challenging. While several inhibitors for PSA Pharmacol Sci 2012;33:623-634. and KLK2, and stimulators for PSA have been 7. Koistinen H and Stenman UH. PSA (Prostate-Specif- developed, their efficacy has not yet been test- ic Antigen) and other Kallikrein-related Peptidases in ed in higher primates or humans. Some of these Prostate Cancer. In: Kallikrein-related peptidases, Vol. 2: Novel cancer-related biomarkers (Eds: Magdolen,V. compounds have been promising in cell culture Sommerhoff, C.P. Fritz, H. Schmitt, M.), De Gruyter and mouse xenograft tumor models. However, 2012, pp. 61-81. the models used so far have several limitations 8. Turk B, Turk du SA and Turk V. Protease signalling: the and do not reflect the complexity of human cutting edge. EMBO J 2012;31:1630-1643. prostate cancer. Furthermore, the specificity of 9. Lopez-Otin C and Bond JS. Proteases: multifunc- the compounds has not been fully elucidated. tional enzymes in life and disease. J Biol Chem 2008;283:30433-30437. Prodrugs that are activated by PSA or KLK2 have been tested clinically in humans with encourag- 10. Quesada V, Ordonez GR, Sanchez LM, Puente XS and Lopez-Otin C. The Degradome database: mammalian ing preliminary results. It is foreseen that these proteases and diseases of proteolysis. Nucleic Acids Res prodrugs, along with vaccines targeting PSA, 2009;37:D239-43. may be the first KLK-based treatment modali- 11. Lawrence MG, Lai J and Clements JA. Kallikreins on ties for prostate cancer. In conclusion, KLKs are steroids: structure, function, and hormonal regulation of prostate-specific antigen and the extended kallikrein lo- attractive targets for prostate cancer treatment, cus Endocr Rev 2010;31:407-446. but much remains to be done before their po- 12. Borgono CA and Diamandis EP. The emerging roles tential can be fully harnessed to treat prostate of human tissue kallikreins in cancer. Nat Rev Cancer cancer. 2004;4:876-890.

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